Name: production of human crispr-engineered car-t cells
Uploaded: 2021-03-15T14:30:00
Description: Watch this Scientific Journal Video about Production of Human CRISPR-Engineered CAR-T Cells at JoVE.com

We acknowledge the Human Immunology Core for providing normal donor T cells and the Flow Cytometry Core at University of Pennsylvania.

Human T cells were procured through the University of Pennsylvania Human Immunology Core, which operates under principles of Good Laboratory Practice with established standard operating procedures and/or protocols for sample receipt, processing, freezing, and analysis conform to MIATA and University of Pennsylvania ethics guidelines.
1. Lentiviral vector production
NOTE: The viral products have been made replication-defective by separation of packaging constructs (Rev...

<ol>
	<li>Kowolik, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+costimulation+provided+through+a+CD19-specific+chimeric+antigen+receptor+enhances+in+vivo+persistence+and+antitumor+efficacy+of+adoptively+transferred+T+cells.">CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells.</a> Cancer Research. 66 (22), 10995-11004 (2006).</li><li>Krause, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-dependent+CD28+signaling+selectively+enhances+survival+and+proliferation+in+genetically+modified+activated+human+primary+T+lymphocytes.">Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes.</a> The Journal of Experimental Medicine. 188 (4), 619-626 (1998).</li><li>Kuwana, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+chimeric+receptor+composed+of+immunoglobulin-derived+V+resions+and+T-cell+receptor-derived+C+regions.">Expression of chimeric receptor composed of immunoglobulin-derived V resions and T-cell receptor-derived C regions.</a> Biochemical and biophysical research Communications. 149 (3), 960-968 (1987).</li><li>Maher, J., Brentjens, R. J., Gunset, G., Rivi&#232;re, I., Sadelain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-lymphocyte+cytotoxicity+and+proliferation+directed+by+a+single+chimeric+TCR&#950;/CD28+receptor.">Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCR&#950;/CD28 receptor.</a> Nature Biotechnology. 20 (1), 70-75 (2002).</li><li>Levine, B. L., Miskin, J., Wonnacott, K., Keir, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+manufacturing+of+CAR-T+cell+therapy.">Global manufacturing of CAR-T cell therapy.</a> Molecular Therapy-Methods &amp; Clinical Development. 4, 92-101 (2017).</li><li>Neelapu, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axicabtagene+ciloleucel+CAR-T-cell+therapy+in+refractory+large+B-cell+lymphoma.">Axicabtagene ciloleucel CAR-T-cell therapy in refractory large B-cell lymphoma.</a> New England Journal of Medicine. 377 (26), 2531-2544 (2017).</li><li>Porter, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+persist+and+induce+sustained+remissions+in+relapsed+refractory+chronic+lymphocytic+leukemia.">Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.</a> Science Translational Medicine. 7 (303), 139 (2015).</li><li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+for+sustained+remissions+in+leukemia.">Chimeric antigen receptor T cells for sustained remissions in leukemia.</a> New England Journal of Medicine. 371 (16), 1507-1517 (2014).</li><li>Melero, I., Rouzaut, A., Motz, G. T., Coukos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+and+NK-cell+infiltration+into+solid+tumors:+a+key+limiting+factor+for+efficacious+cancer+immunotherapy.">T-cell and NK-cell infiltration into solid tumors: a key limiting factor for efficacious cancer immunotherapy.</a> Cancer Discovery. 4 (5), 522-526 (2014).</li><li>Mohammed, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+chimeric+antigen+receptor-modified+T+cell+function+by+reversing+the+immunosuppressive+tumor+microenvironment+of+pancreatic+cancer.">Improving chimeric antigen receptor-modified T cell function by reversing the immunosuppressive tumor microenvironment of pancreatic cancer.</a> Molecular Therapy. 25 (1), 249-258 (2017).</li><li>Moon, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifactorial+T-cell+hypofunction+that+is+reversible+can+limit+the+efficacy+of+chimeric+antigen+receptor-transduced+human+T+cells+in+solid+tumors.">Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors.</a> Clinical Cancer Research. 20 (16), 4262-4273 (2014).</li><li>Beatty, G. L., O'Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+for+the+treatment+of+solid+tumors:+defining+the+challenges+and+next+steps.">Chimeric antigen receptor-modified T cells for the treatment of solid tumors: defining the challenges and next steps.</a> Pharmacology &amp; Therapeutics. 166, 30-39 (2016).</li><li>Stadtmauer, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-engineered+T+cells+in+patients+with+refractory+cancer.">CRISPR-engineered T cells in patients with refractory cancer.</a> Science. 367 (6481), (2020).</li><li>MacLeod, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+a+CD19+CAR+into+the+TCR+alpha+chain+locus+streamlines+production+of+allogeneic+gene-edited+CAR-T+cells.">Integration of a CD19 CAR into the TCR alpha chain locus streamlines production of allogeneic gene-edited CAR-T cells.</a> Molecular Therapy. 25 (4), 949-961 (2017).</li><li>Ren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+editing+to+generate+universal+CAR-T+cells+resistant+to+PD1+inhibition.">Multiplex genome editing to generate universal CAR-T cells resistant to PD1 inhibition.</a> Clinical Cancer Research. 23 (9), 2255-2266 (2017).</li><li>Ure&#241;a-Bail&#233;n, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+technology:+towards+a+new+generation+of+improved+CAR-T+cells+for+anticancer+therapies.">CRISPR/Cas9 technology: towards a new generation of improved CAR-T cells for anticancer therapies.</a> Briefings in Functional Genomics. 19 (3), 191-200 (2020).</li><li>Li, C., Mei, H., Hu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+explorations+of+CRISPR/Cas9+in+CAR-T-cell+therapy.">Applications and explorations of CRISPR/Cas9 in CAR-T-cell therapy.</a> Briefings in Functional Genomics. 19 (3), 175-182 (2020).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-programmed+genome+editing+in+human+cells.">RNA-programmed genome editing in human cells.</a> Elife. 2, 00471 (2013).</li><li>Schluns, K. S., Kieper, W. C., Jameson, S. C., Lefran&#231;ois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-7+mediates+the+homeostasis+of+naive+and+memory+CD8+T+cells+in+vivo.">Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo.</a> Nature Immunology. 1 (5), 426-432 (2000).</li><li>Prlic, M., Lefrancois, L., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+choices:+regulation+of+memory+CD8+T+cell+generation+and+homeostasis+by+interleukin+(IL)-7+and+IL-15.">Multiple choices: regulation of memory CD8 T cell generation and homeostasis by interleukin (IL)-7 and IL-15.</a> The Journal of Experimental Medicine. 195 (12), 49-52 (2002).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+(CAR-T)+cells+expanded+with+IL-7/IL-15+mediate+superior+antitumor+effects.">Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects.</a> Protein &amp; Cell. 10 (10), 764-769 (2019).</li><li>Hultquist, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9+genome+engineering+of+primary+CD4++T+cells+for+the+interrogation+of+HIV-host+factor+interactions.">CRISPR-Cas9 genome engineering of primary CD4+ T cells for the interrogation of HIV-host factor interactions.</a> Nature Protocols. 14 (1), 1-27 (2019).</li><li>Brinkman, E. K., Chen, T., Amendola, M., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucleic Acids Research. 42 (22), 168 (2014).</li></ol>

Here we describe approaches to gene edit CAR-T cells using CRISPR Cas9 technology and manufacture products to further test for function and efficacy. The above protocol has been optimized for performing CRIPSR gene editing in primary human T cells combined with engineering T cells with chimeric antigen receptors. This protocol allows high knockout efficiency with minimal donor-to-donor variability. Modification using CRISPR can improve both the efficacy and safety of CAR-T cells by eliminating receptors that inhibit T ce...

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the field of adoptive cell therapies and cancer immunotherapy. CAR-T-cells are engineered T-cells expressing a synthetic immune receptor that combines an antigen-specific single chain antibody fragment with signaling domains derived from the TCRzeta chain and costimulatory domains necessary and sufficient for T-cell activation and co-stimulation1,2,3,4. The manufacturing of CAR-T cells starts by extracting the patient&#39;s own T-cells, followed by ex vivo viral transduction of the CAR module and expansion of the CAR-T cell product with magnetic beads that function as artificial antigen presenting cells5. Expanded CAR-T cells are re-infused into the patient where they can engraft, eliminate target tumor cells and even persist for multiple years post infusion6,7,8. Although CAR-T cell therapy has resulted in remarkable response rates in B-cell malignancies, clinical success for solid tumors has been challenged by multiple factors including poor T-cell infiltration9, an immunosuppressive tumor microenvironment10, antigen coverage and specificity, and CAR-T cell dysfunction11,12. Another limitation of current CAR-T cell therapy includes the use of autologous T-cells. After multiple rounds of chemotherapy and high tumor burden, CAR-T cells can be of poor quality as compared to allogeneic CAR-T products from healthy donors in addition to the time and expense associated with manufacturing of autologous CAR-T cells. Gene-editing of the CAR-T cell product by CRISPR/Cas9 represents a new strategy to overcome current limitations of CAR-T cells13,14,15,16,17.
CRISPR/Cas9 is a two component system that can be used for targeted genome editing in mammalian cells18,19. The CRISPR-associated endonuclease Cas9 can induce site-specific double-strand breaks guided by small RNAs through base-pairing with the target DNA sequence20. In the absence of a repair template, double-strand breaks are repaired by the error prone nonhomologous end joining (NHEJ) pathway, resulting in frameshift mutations or premature stop codons through insertion and deletion mutations (INDELs)19,20,21. Efficiency, ease of use, cost-effectiveness and the ability for multiplex genome editing make CRISPR/Cas9 a powerful tool to enhance the efficacy and safety of autologous and allogeneic CAR-T cells. This approach can also be used to edit TCR directed T cells by replacing the CAR construct with a TCR. Additionally, allogeneic CAR-T cells that have limited potential to cause graft versus host disease can also be generated by gene editing the TCR, b2m, and HLA locus.
In this protocol, we show how CRISPR-engineering of T cells can be combined with viral vector mediated delivery of the CAR-Transgene to generate genome-edited CAR-T cell products with enhanced efficacy and safety. A schematic diagram of the entire process is shown in Figure 1. Using this approach, we have demonstrated high-efficiency gene knockout in primary human CAR-T cells. Figure 2A describes in detail the timeline of each step for editing and manufacturing T cells. Strategies for guide RNA design and knockout validation are also discussed to apply this approach to various target genes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4D-Nucleofactor Core Unit</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>4D-Nucleofactor X-Unit</td>
			<td>Lonza</td>
			<td>AAF-1002X</td>
			<td></td>
		</tr>
		<tr>
			<td>Accuprime Pfx Supermix</td>
			<td>ThermoFisher</td>
			<td>12344040</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Optima XPN ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-human CD3 Antibody</td>
			<td>Biolegend</td>
			<td>317322</td>
			<td>Clone OKT3</td>
		</tr>
		<tr>
			<td>BV711 Anti-human PD1</td>
			<td>Biolegend</td>
			<td></td>
			<td>Clone EH12.2H7</td>
		</tr>
		<tr>
			<td>Cas9-Electroporation enhancers</td>
			<td>IDT</td>
			<td>1075915</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3/CD28 Dynabeads</td>
			<td>ThermoFisher</td>
			<td>40203D</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation Kit</td>
			<td>StemCell technologies</td>
			<td>15062</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8+ T cell isolation Kit</td>
			<td>StemCell technologies</td>
			<td>15063</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 0.45 micron vacuum filter/bottle</td>
			<td>Corning</td>
			<td>430768</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning T150 cell culture flask</td>
			<td>Millipore Sigma</td>
			<td>CLS430825</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Millipore Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAeasy Blood and Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag Magnet</td>
			<td>ThermoFisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>ThermoFisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>huIL-15</td>
			<td>PeproTech</td>
			<td>200-15</td>
			<td></td>
		</tr>
		<tr>
			<td>huIL-7</td>
			<td>PeproTech</td>
			<td>200-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>ThermoFisher</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Gel and PCR cleanup</td>
			<td>Takara</td>
			<td>740609.25</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>ThermoFisher</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>P3 Primary cell 4D-nucleofactor X Kit L</td>
			<td>Lonza</td>
			<td>V4XP-3024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin-Glutamine</td>
			<td>ThermoFisher</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>pTRPE expression Plasmid</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Mouse FMC63 scFv Monoclonal Antibody, (R19M), PE</td>
			<td>CytoArt</td>
			<td>200105</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>ThermoFisher</td>
			<td>12633012</td>
			<td></td>
		</tr>
		<tr>
			<td>sgRNA</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spy Fi Cas9</td>
			<td>Aldevron</td>
			<td>9214</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Viral packaging mix</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X-Vivo-15 Media</td>
			<td>Lonza</td>
			<td>BE02-060F</td>
			<td></td>
		</tr>
	</tbody>
</table>

production of human crispr-engineered car-t cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

We describe here a protocol to genetically engineer T cells, that can be used to generate both autologous and allogeneic CAR-T cells, as well as TCR redirected T cells.
Figure 1 provides a detailed description of the stages involved in the process of manufacturing CRISPR edited T cells. The process begins by designing sgRNA to the gene of interest. Once the sgRNA are designed and synthesized they are then used to make RNP complexes with the appropriate Cas protein...

Watch this Scientific Journal Video about Production of Human CRISPR-Engineered CAR-T Cells at JoVE.com

Production of Human CRISPR-Engineered CAR-T Cells

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

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